Proteins have a diverse range of physical properties and perform a wide range of biological functions, including enzymatic catalysis, transport and storage, mechanical functions, movement, protection, information processing, and the like. Although proteins are manufactured naturally by living organisms to carry out these functions, many proteins can now be manufactured in large quantities using biological engineering technologies. Manufactured proteins have become important commercial products for treating disease and illness in human being and animal patients. For example, the protein insulin is used for treating patients with diabetes. Other commercially important proteins include tissue plasminogenactivator (TPA), erythropoieton (EPO), human growth hormone (hGH), interleukin II, and the like.
All proteins are composed of a linear sequence of amino acid residues. The amino acid residues are linked together by peptide bonds occurring between the amino group of one amino acid and the carboxyl group of the preceding amino acid. Molecules which are composed of a sequence of amino acid residues, such as proteins, are known generally as peptides. Proteins are a class of peptides referred to as polypeptides and typically comprise a sequence of fifty or more amino acid residues. As such, proteins are extremely large biological molecules having molecular weights typically exceeding 5000 amu. For example, insulin is characterized by a sequence of 51 amino acid residues and a molecular weight of 5808 daltons. As another example, human growth hormone is characterized by a sequence of 191 amino acids and a molecular weight of 22,125 daltons.
Each kind of protein has a unique sequence of amino acids. Additionally, the relative abundances of the various kinds of amino acid residues in a protein also tends to be unique. As such, the amino acid sequence and relative abundances of the amino acid residues are fingerprints of a protein molecule. Generally, the function or functions of a protein are often extremely dependent upon such fingerprint characteristics. Even minor changes in such characteristics may destroy the function or functions of a protein molecule. Examples of possibly deleterious changes include omission of an amino acid residue in a sequence, inclusion of an extra amino acid residue in sequence, a change in the amino acid order of a sequence, substitution of an incorrect amino acid residue in place of the desired amino acid residue, undesirable oxidation of one or more functional groups of one or more amino acid residues, N-terminal modifications, unintended addition, deletion, or modification of side chains, unintended cleavage, unintended hydroxylation, and the like.
Accordingly, when studying, developing, or manufacturing protein products, it is critical that the protein product under consideration is being, or has been, manufactured correctly and consistently. It is essential, therefore, to be able to qualitatively and quantitatively compare production material to product standards. Because of the enormous size of protein molecules, however, it is not practical to work with an entire protein molecule when performing such qualitative and/or quantitative analysis. Accordingly, it is desirable to first cleave a protein into smaller, more manageable pieces by hydrolysis of the peptide bonds between one or more amino acid residues. The result of such hydrolysis is an admixture comprising one or more peptides and/or one or more individual amino acids, depending upon the hydrolysis conditions that were used.
Proteins can be cleaved into smaller pieces using a variety of techniques, including chemical and enzymatic digestion. Enzymatic digestion is one of the most frequently used techniques for cleaving a protein molecule into smaller, more manageable pieces, because some specific enzymes tend to cleave a protein molecule at extremely specific cleavage sites. For example, the enzyme trypsin cleaves a protein molecule only on the carboxyl side of the amino acid residues of lysine (abbreviation K) and arginine (abbreviation R). As another example, the enzyme chymotrypsin cleaves protein molecules on the carboxyl side of the aromatic amino acid residues phenylalanine, tyrosine, and tryptophan. Theoretically, digestion of two identical protein molecules by the same enzyme activity should yield two identical admixtures of peptide digests. Techniques for carrying out enzymatic digestion are widely known in the art and are generally described in G. Allen, Laboratory Techniques in Biochemistry and Molecular Biology (R. H. Burdon and P. H. Knippenburg; eds.) Vol. 9, Sequencing of Proteins and Peptides, Elsevier Press, 1989.
Unfortunately, even if a protein is cleaved into smaller, more manageable pieces, comparing the resultant pieces of one protein molecule to those of another protein molecule is still a challenging problem. The problem is particularly vexing for qualifying commercial production material in which comparison between production material and product standards is desirably accomplished relatively quickly, because many of the previously known methods for characterizing a protein sufficiently to allow meaningful comparisons to be made are too expensive, too time consuming, too inaccurate, and/or yield too little information about the protein. Meaningful and practical comparisons between production material and product standards has remained on the wish list of the industry for a long time. Accordingly, methods allowing fast, accurate, repeatable, informative, and economical comparisons of protein molecules are needed.